An antibody molecule consists of two polypeptide chains, each of which has an H-chain and L-chain. It is known that each of the H- and L-chains comprises a sequence of domains consisting of about 110 amino acids each and that an L-chain comprises two domains (VL and CL) and an H-chain comprises four domains (VH, CH1, CH2 and CH3). The structural gene (VH gene) encoding a variable region of an H-chain comprises three gene fragments, i.e., V, D and J gene fragments, while the structural gene (VL gene) encoding an L-chain comprises two gene fragments, i.e., J and V gene fragments regardless of whether the L-chain belongs to a λ or κ chain. On such variable regions, an antigen-binding site responsible for the recognition of a specific antigen is formed. Immunoglobulin molecules are widely diversified in their primary structure, even when they are derived from the same animal species and belong to the same class, and particularly contain the hypervariable regions, whose diversity is remarkable. In a hypervariable region, complementarity-determining regions (CDRs), each of which has a 3D structure complementary to a specific antigen molecule and thus determines the specificity of the antibody, and framing regions (FRs), which support the 3D structure of the hypervariable region, are arranged in a mosaic fashion. It has been known that the hypervariable region formed on each of the H- and L-chains comprises three highly variable regions called CDR1, CDR2, and CDR3, which can specifically bind to various antigens as a result of the diversity of amino acid sequences of CDRs.
Usually, an antibody is made by a method of immunizing an animal with a foreign antigen. Accordingly, it has been thought that it is difficult to prepare a specific antibody with an animal against its own substance or a substance that is closely similar to it. Recently, a method for making antibodies using a phage antibody library has been established. This method is an application of displaying an antibody on the surface of phages by fusing an antibody to the coat protein of a filamentous phage. According to this method, a library having a wide variety of antibodies is made by amplifying the genes encoding antibody by PCR, and by displaying it on the phage particles. By this method it is possible to prepare antibodies against a human antigen. A screening method using a phage display library is called a phage display method and has been used for the identification of ligands that specifically bind to various receptors of cells or various antibodies. Many reports have been already published on how to prepare phage antibody libraries, immuno-tube methods using such a phage antibody library, and screening methods such as magnetic bead method (Science, 249:386-390(1990); Methods in Enzymology, 217:228-257(1993)).
Such an antibody library including an enormous repertory described above is an excellent technique that can save immunological operations and time for screening, can be widely applicable for the preparation of various antibodies, and simplify screening. However, an antibody library that has been prepared so far is problematic because of contamination of a repertory that cannot be expressed and instability. In an antibody library that has been prepared so far, amplification rate of individual clones are often varied, and thus it is difficult to maintain an enormous repertory. Methods for other library preparations were reported, by which a repertory involved in the production of antibodies in a spleen or bone marrow cells is used or random amino acid sequences are integrated in immunoglobulin genes to obtain a repertory with 1012 or more antibodies (see, for example, J. Molecular Biology, 248:97-105, 1995). With these methods, however, the contamination of a repertory which cannot be expressed, such as genes that encode non-functional antibodies, which have occurred by insertion of a translation termination codon or influence to high-order structures of antibody molecules, or instability of the library cannot be avoided.
On the other hand, VL-VH shuffling via E. coli in which shuffling is made in an sc Fv (single-chain Fragment variable) library to increase the size of its repertory, i.e. VH-gp3 fragments are shuffled (Nature Biotech., 18:75-80, 2000), preparation of a library of recombinant polynucleotides derived from single or double stranded parental DNA templates as various starting materials (PCT Japanese Translation Patent Publication No. 2001-516215), and method for producing catalytic antibodies that are displayed on phages comprising processes for preparing a library of DNA fragments encoding an antibody domain, inserting the domain-encoded fragments into a phage expression vector, and isolating the catalytic antibodies (PCT Japanese Translation Patent Publication No. 10-507341). However, there has never been reported a useful single-chain antibody library having a repertory of 1011 or more antibodies, for example, 1012 antibodies. In addition, no method has existed for selectively producing a single chain antibody comprising a VH and VL regions obtained by shuffling.